Functional Expression of Bovine Opsin in the Methylotrophic YeastPichia pastoris

Abstract

The methylotrophic yeastPichia pastoriswas examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from theP. pastorisalcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal. Quantitative–competitive PCR analysis of a stable yeast transformant showed that one copy of the opsin gene was integrated into the yeast genome. The expression level in this transformant corresponded to ∼0.3 mg of opsin per liter of cell culture (A600= 1.0). Sucrose density sedimentation analysis indicated that the opsin was associated exclusively with the membrane fraction. Similar to retinal opsin,P. pastoris-expressed opsin migrated as a single band of ∼37 kDa on SDS–PAGE and showed high mannose N-glycosylation. A portion of the expressed opsin (∼4–15%) reacted with 11-cis-retinal to form the rhodopsin chromophore (λmax500 nm), and after purification showed ground and excited state spectral characteristics indistinguishable from those of the native pigment. Further, the metarhodopsin-II-mediated G-protein-activating potential of yeast expressed rhodopsin was similar to that of native rhodopsin. These results show thatP. pastoriscells have the capacity to functionally express bovine opsin.